1. Field of the Invention
The invention is concerned with single measurement Excitation Emission Matrix (EEM) spectroscopy using an array of light emitting diodes (LED). The array of LEDs is focused into a sample cuvette, creating spacially separated excitation spots.
2. Description of the Related Prior Art
Fluorescence excitation emission matrix (EEM) spectroscopy has long been known as a powerful method for complex mixture analysis. (See Hershberger, L. W.; Callis, J. B.; Christian, G. D.; Anal. Chem., 1981, 53, 971–975; Skoropinski, B.; Callis, J. B.; Danielson, J. D. S.; Christian G. D.; 1986, Anal. Chem., 58, 2831–2839; and Jalkian, R.; Denton, B.; Proc. SPIE 1989, 1054, 91–102.)
The ability to easily collect full emission spectra for several excitation wavelengths, has generally required expensive and complex instrumentation. The multi-way characteristics of EEM data enable the extraction of the salient chemical features. As a consequence, mathematical resolution of analytes is possible, even in the presence of unknown interferences, which is known as the second-order advantage. (See Booksh, K. S.; Kowalski, B. R.; Anal. Chem., 1994, 66, A782–A791.)
The full range of molecules and dyes which fluoresce under long wavelength UV (370 nm) through near infra-red (NIR) wavelength (980 nm) excitation can be used with this inexpensive and simple to construct EEM system. The potential applications of upper UV, visible, and NIR fluorescence include fluorescence of dyes, larger PAHs (anthracene, chrysene, benzopyrene, perylene, etc.), humic materials, (See Baker, A.; Environ. Sci. Tech., 2002, 36, 7, 1377–1382; and Del Castillo, C. E.; Coble, P .G.; Morell, J. M.; Lopez, J. M.; Corredor, J. E.; Mar. Chem., 1999, 66, 35–51.) chlorophylls from plants (See Moberg, L.; Robertsson, G.; Karlberg, B.; Talanta, 2001, 54, 161–170.) and algae (See Henrion, R.; Henrion, G.; Bobme, M.; Behrendt, H.; Fresen. J. Anal. Chem., 1997, 357, 522–526.) and NIR fluorescence from bacteriochlorophylli (See Albrecht-Buehler, G.; Exp. Cell Res., 1996, 236, 43–50) in certain bacteria. Other applications include environmental dye tracers (See. Smart, P. L.; Laidlaw, I. M. S.; Water Resour. Res., 1977, 13 (1), 15–33; and Lyons, R. G.; J. Hydrol., 1993, 152 (1–4), 13–29) and porphyrin fluorescence. (See Ricchelli. F.; Gobbo, S.; J. Photochem. Photobiol. B, 1995, 29 (1), 65–70.) Fluorescence of porphyrins and derivatives can be used for detection of certain cancers using patient sera analysis (See Aiken, J. H.; Huie, C. W.; Terzian, J. A.; Anal. Lett., 1994,27(3), 511–521.), and as markers for heavy metal poisoning through urine analysis. (See Bowers, M. A.; Aicher, L. D.; Davis, H. A.; Woods, J. S.; J. Lab. Clin. Med., 1992, 120 (2), 272–281; and Ng, J. C.; Qi, L. X.; Moore, M. R.; Cell. Mol. Biol., 2002, 48 (1), 111–123.)
The application of LED array excitation is well suited to fluorescence in situ hybridization (FISH) of cell and bacterial suspensions. The true advantage, in this arena, with the LED-EEM system is for multiplexed fluorescence in-situ hybridization (M-FISH) (See Henegariu, O.; Bray-Ward, P.; Ward, D. C.; Nat. Biotechnol., 2000, 18 (3), 345–348.), where many dyes (5–10) are used to simultaneously detect various cell or bacterial types using specific oligo-nucleotides. Furthermore, the possibility of multiplexing FISH-based genome analysis is very attractive. (See Fauth, C.; Speicher, M. R.; Cytogenet. Cell Genet., 2001, 93 (1–2), 1–10; Speel, E. J. M.; Histochem. Cell Biol., 1999, 112 (2), 89–113; and Szuhai, K.; Bezrookove, V.; Wiegant, J.; Vrolijk, J.; Dirks, R. W. Rosenberg, C.; Raap, A. K.; Tanke, H. J.; Gene. Chromosom. Canc., 2000, 28 (1), 92–97.) The availability of full emission spectra at multiple excitation wavelengths will allow better characterization of FISH dyes. This can be achieved using readily available fluorophores, without resorting to expensive designer dyes which absorb maximally at a common wavelength (typically 488 nm—argon ion laser) and emit in well separated regions for simplified detection. Future advances in LED technology may result in lower UV wavelengths being made available, thus extending the range of applications.